Tobacco smoking is a leading cause of preventable deaths in the United States and worldwide. Understanding why tobacco smoking is highly addictive and identifying the mechanisms underlying addiction would be of tremendous benefit in developing new therapies for smoking cessation needed for preventing addiction. Nicotine is the primary agent in tobacco leading to addiction. Addiction is initiated by nicotine binding to nicotinic acetylcholine receptors (nAChRs) in the brain. High-affinity nAChRs, mainly 42 nAChRs in the brain's reward areas, mediate the reinforcing effects of nicotine. The only long-lasting effect, i.e., greater than a few minutes, of nicotine directly on nAChRs is a phenomenon called nicotine-induced upregulation of nAChRs (upregulation). We have demonstrated that nAChR upregulation is much more complex than originally assumed consisting of multiple processes that occur at very different rates and are caused by different mechanisms. One goal of the proposed research is to characterize in more detail the two components of nAChR upregulation that we have recently discovered. In the first Aim, we will examine how nicotine exposure increases nAChR endocytosis and recycling of 42 nAChRs in neurons and heterologous cells. In the second Aim, we will test in more detail how nicotine exposure regulates the glycosylation of 42 nAChRs. Another goal of the proposed research is to examine how nAChR ligands and smoking cessation agents affect the different components of upregulation. These experiments will be performed in the third Aim.